(1) Biosynthesis of Polypeptides (Proteins)
In the biosynthesis of polypeptides, the stage during which a polypeptide is produced from a template mRNA is called translation. A set of three consecutive nucleotides in mRNA is called codon. Each codon corresponds to one amino acid, but the three codons UAA, UAG and UGA have no corresponding amino acid and signal a termination of polypeptide synthesis so that they are called stop codons. On the other hand, the first codon AUG in the translation of mRNA, which signals an initiation of polypeptide synthesis, is called start codon. Each set of three nucleotides following the start codon corresponds to one amino acid.
In translation, it is important that a cognate amino acid should be correctly assigned to the tRNA having the function of reading each codon on the mRNA used as a template for polypeptide synthesis. Chemically, translation is achieved via ester linkage of an amino acid to the 3′-end of a specific tRNA at the carboxyl group. For example, methionine binds to an initiator tRNA (or tRNAfMet) corresponding to the start codon, then its amino group is formylated (attached to a —COH group) to form N-formylmethionine (FIG. 1A: translation initiation in nature). Thus, the prokaryotic initiator tRNA (fMet-tRNAfMet) is synthesized. AUG is the only codon corresponding to methionine.
The AUG codon is also important as a start codon that signals the ribosome to “initiate” protein translation from mRNA. The ribosome is a protein synthesizer consisting of an assembly of 50 or more ribosomal proteins and several RNA molecules (rRNA), which reads genetic information of mRNA to catalyze amino acid polymerization. The ribosome is very similar in structure and function between eukaryotes and prokaryotes, and forms a complex of a molecular mass exceeding several million daltons consisting of one large subunit and one small subunit.
The process of initiating the synthesis of prokaryotic-derived polypeptides involves a number of steps in which proteins called initiation factors (IFs) participate. First, an initiator tRNA aminoacylated with methionine is converted into N-formylmethionine-tRNA by a methionine tRNA formyltransferase (MTF) and binds to an initiation factor. Then, the ribosomal small subunit binds to this initiation factor/N-formylmethionine-tRNA conjugate, and the resulting complex binds to the ribosome-binding site (SD sequence) on mRNA. When this complex finds a start signal (AUG codon), the large subunit binds to it. At the same time, the initiation factor dissociates from the complex, and a ribosome/initiator tRNA complex remains on mRNA. Initiation of translational peptide synthesis occurs through a correct sequence of these steps so that the synthesized product normally has a formylmethionine at the N-terminus.
Then, the ribosome translates codons one after another while moving along mRNA toward the 3′-end, and adds an amino acid to the end to be elongated of the polypeptide by using tRNA. The amino acid added to the end to be elongated of the polypeptide chain is chosen by complementary base pairing between the anticodon of the tRNA molecule to which the amino acid is bound and the subsequent codon of the mRNA strand. In this manner, amino acids corresponding to the codons of mRNA are joined by peptide linkages one after another so that polypeptide synthesis proceeds.
(2) Amino Acid Specificity of tRNA
As already noted, it is tRNA that plays a role as an adapter assigning the codons of mRNA as genetic information to amino acids. Each tRNA acts as an adapter by binding to (aminoacylating) an amino acid specific to it. As a crucial factor for translation accuracy, a strict correspondence is required between the anticodon of each tRNA and an amino acid. However, tRNA and the anticodon do not directly choose an amino acid, but an aminoacyl-tRNA synthetase (ARS) shows specificity to each amino acid, and each tRNA molecule specifically recognizes its cognate ARS and is aminoacylated to accept a correct amino acid. In other words, the amino acid specificity of tRNA in vivo is maintained by specific molecular recognition between tRNA and ARS.
On the other hand, methods for mischarging tRNA with a substance other than the amino acid that should be originally accepted were proposed by artificially changing the specific correspondence among the three members, i.e., tRNA, ARS, and amino acid. One of such methods uses an ARS ribozyme developed by us via in vitro molecular evolution, which catalyzes tRNA acylation reaction (also known as acylase RNA or commonly called “Superflexizyme”). Superflexizyme is characterized in that it allows aminoacylation using any tRNA anti any amino acid. In other words, it allows any tRNA to bind to any amino acid at will. This is very useful for e.g., translationally synthesizing a polypeptide containing an unnatural (unusual) amino acid (patent documents 1, 2, non-patent documents 1, 2, 3, 4).
(3) Cell-free Synthesis
Cell-free polypeptide synthesis is to synthesize a polypeptide in vitro in a genetic information translation system formed of a cytoplasmic extract in an artificial container. Cell-free synthesis using no living organism is free from physiological constraints in vivo, and expected to achieve high-throughput polypeptide synthesis from genes and to dramatically enlarge the range of amino acid sequences that can be synthesized. In principle, it is thought that polypeptides consisting of any amino acid sequence can be synthesized in vitro at will only in the presence of genetic information in cell-free polypeptide synthesis systems unless the catalytic function of the translation enzyme system is disturbed. Moreover, unnatural amino acids not occurring in vivo can also be used if they can be successively assigned to genetic information.
(4) Peptidyl Compounds Having an Unnatural Structure at the N-termini
Naturally derived peptidyl compounds sometimes contain a structurally unique amino acid attached to the N-termini. In the examples shown in FIG. 2, Somamides A [FIG 2A] has a hexyl group and Factor A (A54556 complex) [FIG. 2B] has a 2,4,6-heptatrienyl group exist at the N-termini. Many of neuropeptides found in vivo have a pyroglutamic structure at the N-termini.
These molecules are long peptides that are inevitably expensive because they are difficult to chemically synthesize or they are synthesized at low yields. If one desires to discover a drug by synthesizing a wide variety of mimetic peptides in parallel (library construction), additional molecules encoding the peptide sequences should be chemically conjugated onto beads or peptide molecules, which further adds technical complexity. Moreover, if the peptide library has been exhausted, a completely new library should be synthesized again. On the other hand, no case has been reported in which such a polypeptide was successfuly synthesized through an artificial translation system regardless of whether it is a living cell system or a cell-free system. Thus, if a technique capable of translationally synthesizing these unique peptide molecules by allowing a template mRNA to encode a sequence were developed, a significant technical progress would be made.
References:
Patent document 1: JPA No. 2003-514572.
Patent document 2: JPA No. 2005-528090.
Non-patent document 1: H. Murakami, H. Saito, and H. Suga (2003) “A versatile tRNA aminoacylation catalyst based on RNA” Chemistry & Biology, Vol. 10, 655-662.
Non-patent document 2: Tanpakushitsu Kakusan Kouso (2003) Vol. 48, No. 11, pp. 1511-1518.
Non-patent document 3: Jikkenn Igaku (2004) Vol. 22, No. 17, pp. 184-189.
Non-patent documents 4: H. Murakami, A. Ohta, H. Ashigai, H. Suga (2006) “The flexizyme system: a highly flexible tRNA aminoacylation tool for the synthesis of nonnatural peptides” Nature Methods 3, 357-359.